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WO2005085426A1 - Milieu en vue d'une différenciation exempte d'alimentateur et procédé de différenciation exempt d'alimentateur à partir d'une cellule souche embryonnaire de primate - Google Patents

Milieu en vue d'une différenciation exempte d'alimentateur et procédé de différenciation exempt d'alimentateur à partir d'une cellule souche embryonnaire de primate Download PDF

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Publication number
WO2005085426A1
WO2005085426A1 PCT/JP2005/003474 JP2005003474W WO2005085426A1 WO 2005085426 A1 WO2005085426 A1 WO 2005085426A1 JP 2005003474 W JP2005003474 W JP 2005003474W WO 2005085426 A1 WO2005085426 A1 WO 2005085426A1
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cells
feeder
free
medium
embryonic stem
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Japanese (ja)
Inventor
Hong Zhang
Kumiko Saeki
Akira Yuo
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Mitsubishi Tanabe Pharma Corp
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Tanabe Seiyaku Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to a culture medium for differentiating primate embryonic stem cells into blood cells or vascular endothelial cells, a culture system for shunting, a method for differentiating blood cells or vascular endothelial cells, blood cells or blood vessels.
  • the present invention relates to a method for producing endothelial cells and cells obtained thereby. More specifically, the present invention provides a medium for feeder-free differentiation from primate embryonic stem cells to blood cells or vascular endothelial cells, a culture system for feeder-free differentiation, a feeder-free differentiation method, a blood cell or
  • the present invention relates to a method for producing vascular endothelial cells and cells obtained thereby.
  • Embryonic stem cells were first established in mice in the 1980s, and have been mainly used to create disease model animals. On the other hand, there is a possibility that it can be used as a basic material in organ creation in the fields of regenerative medicine and transplantation medicine as a universal cell that divides into various cells. Therefore, establishment of human embryonic stem cells has been awaited. Then, in 1998, human embryonic stem cells were established in the United States. Research has been conducted on the division of embryonic stem cells obtained from experimental animal models such as mice (see Patent Document 1).
  • NK cells which are the basis of immunity, attack foreign substances invading the body, that NK cells are effective against cancer, etc., and that hematopoietic stem cells, etc. are effective in leukemia, etc.
  • hematopoietic stem cells are of great medical importance because of their fibroplastic plasticity and their ability to transdifferentiation into cells required for various diseases.
  • a large amount of blood cells can be produced from embryonic stem cells, for example, by factory production, the contribution to medical treatment is immense.
  • red blood cells and the like used for blood transfusion and the like can be obtained by the technique of dividing embryonic stem cell power, the problem of contamination such as AIDS and hepatitis C in blood donation can be solved.
  • neutrophils can be used for the immune system damaged by chemotherapy for cancer. The transfusion of leukocytes containing these can enhance immunity with reduced function, thus solving problems such as hospital-acquired infections.
  • blood cells have a profound effect on medicine and the medical industry, as they also lead to enhanced natural healing powers.
  • Non-Patent Document 1 a method of co-culturing embryonic stem cells using OP9 cells, which is a stromal cell line, as a feeder has been attempted (eg, , Non-Patent Document 1).
  • the Ca ⁇ mow fraction I ⁇ method after differentiated into mesodermal cells by culturing in seeding the embryonic stem cells on OP9 cells or embryoid bodies from embryonic stem cells; form (embry 0 id EB) After differentiation into mesodermal cells, the mesodermal cells are dissociated by trypsin treatment or the like, seeded again on OP9 cells, and cultured to separate blood cells.
  • Patent Document 1 International Publication No. 99Z20741 pamphlet
  • Non-Patent Document 1 Toru Nakano (Nakano T.) et al., ⁇ In vitro development of primitive and definitive erythrocytes from different precursors '', Science, Vol. 272, pp. 722-724 (1996) Disclosure of the Invention
  • a first aspect of the present invention is to differentiate primate embryonic stem cells into blood cells or vascular endothelial cells substantially without contaminating foreign animal cells, infecting foreign animal-derived viruses, and the like.
  • a second aspect of the present invention is to perform differentiation of primate embryonic stem cell blood cells into blood cells substantially without contaminating heterologous animal cells, transmitting a virus derived from a xenogeneic animal, and the like.
  • An object of the present invention is to provide a feeder-free culture system for one-sided feeding that enables the differentiation from blood stem cells into blood cells under high and strict control.
  • the third aspect of the present invention relates to contamination of foreign animal cells, Primate embryonic stem cell power without substantially involving staining etc., and at least the ability to differentiate into blood cells and to differentiate primate embryonic stem cells into blood cells with high and strict control.
  • a fourth aspect of the present invention is to differentiate primate embryonic stem cells into vascular endothelial cells substantially without contaminating foreign animal cells, infecting with foreign animal-derived viruses, and the like.
  • the fifth aspect of the present invention relates to a method for producing blood cells that enables at least one of obtaining high-purity blood cells, obtaining blood cells with excellent reproductivity over a long period of time, and the like. Is to provide a law.
  • a sixth aspect of the present invention is to provide a method for producing vascular endothelial cells, which enables high-purity vascular endothelial cells to be obtained.
  • a seventh aspect of the present invention is to provide a blood cell or a vascular endothelial cell exhibiting high purity and uniform properties.
  • An eighth aspect of the present invention is to provide a method for producing finger-like cells, which can efficiently obtain large quantities of finger-like cells that can be divided into blood cells, vascular endothelial cells, and the like.
  • a ninth aspect of the present invention is to provide finger-like cells capable of supplying blood cells, vascular endothelial cells, and the like suitable for clinical purposes and the like. Other objects of the present invention will be apparent from the description in this specification and the like.
  • the gist of the present invention is:
  • a feeder-free differentiation medium comprising a stromal cell conditioned medium
  • the site force-in is vascular endothelial growth factor, bone morphogenetic protein 4, stem cell factor, Fit
  • the stromal cells are irradiated stromal cells, the (1) one (4) medium without feeder according to any one of (1),
  • a feeder-free differentiation medium which is obtained by performing a process comprising:
  • ⁇ ' irradiating the cells obtained in the step A) with radiation, and culturing the obtained cells in a medium suitable for maintaining stromal cells and embryonic stem cells;
  • a feeder-free differentiation medium according to any one of (1) to (5), which is obtained by performing a process comprising:
  • a feeder-free fractionating medium comprising the feeder-free differentiation medium according to any one of [1] to [7] and a culture vessel coated with extracellular matrix.
  • a primate animal embryo wherein the primate animal embryonic stem cells are cultured under a feeder-free medium in the feeder-free differentiation medium according to any one of [1] to [7].
  • Feeder-free differentiation from stem cells to blood cells [12] Culturing primate embryonic stem cells in a feeder-free differentiation medium according to any one of [1] and [7] above, under a feeder-free condition, in a culture vessel coated with an extracellular matrix Feeder-free differentiation of primate embryonic stem cells into blood cells as described in [11] above, thereby generating finger-like cells and further culturing to produce blood cells.
  • a primate embryonic stem cell is cultured in a feeder-free differentiation medium according to any one of [1] and [7] above, under a feeder-free condition, in a culture vessel coated with an extracellular matrix. Then, finger-like cells are generated, and the finger-like cells are transferred to a culture vessel containing a new medium and coated with extracellular matrix, and further cultured to generate vascular endothelial cells.
  • a feeder-free differentiation method from primate embryonic stem cells to vascular endothelial cells
  • a primate embryonic stem cell is cultured in a culture vessel coated with an extracellular matrix in a feeder-free differentiation medium according to any one of [1] and [7] above and below a feeder-free. And then differentiating the embryonic stem cells into blood cells, and then detaching and isolating the blood cells,
  • the feeder-free differentiation medium described in any one of (1) to (7) above is added to the culture vessel after blood cell detachment, and further cultured under a feeder-free cell.
  • [17] Culturing a primate embryonic stem cell in a culture vessel coated with an extracellular matrix in a feeder-free medium for differentiation according to any one of [1] to [7] above and without a feeder Then, finger-like cells are generated, and the finger-like cells are transferred to a culture vessel containing a fresh medium and coated with extracellular matrix, and further cultured, whereby the embryonic stem cell capillar vascular endothelium is cultured.
  • a method for producing vascular endothelial cells which comprises differentiating the cells into cells and isolating the vascular endothelial cells;
  • a primate embryonic stem cell is cultured in a culture vessel coated with an extracellular matrix in a feeder-free differentiation medium according to any one of (1) to (7) above, under a feeder-free condition.
  • a primate animal embryonic stem cell which is substantially free from heterogeneous animal cell contamination, xenogeneic virus infection, etc. in the absence of xenogeneic animal cells. It has an excellent effect that blood cells can be separated. Further, according to the feeder-free medium for fractionation of the present invention, differentiation with excellent reproductivity can be performed, and further, blood cells or vascular endothelial cells can be obtained with high efficiency and high purity. !, Has excellent effect.
  • ADVANTAGE OF THE INVENTION According to the culture system for feeder-free fractionation of the present invention, the primate embryo which is substantially free from contamination of foreign animal cells, infection of foreign animal-derived virus, etc.
  • the separation can be carried out substantially without the contamination of foreign animal cells, infection of foreign animal-derived viruses, and the like. It has an excellent effect that it can be differentiated from primate embryonic stem cells into blood cells with high and strict control.
  • the method of feeder-free differentiation of primate embryonic stem cells into vascular endothelial cells of the present invention does not substantially involve the contamination of foreign animal cells, infection with a virus derived from a foreign animal, or the like.
  • the method for producing blood cells of the present invention it is possible to obtain high-purity blood cells that are long-lasting, have excellent reproductivity, and have an excellent effect. Further, according to the method for producing a vascular endothelial cell of the present invention, an excellent effect that high-purity vascular endothelial cells can be obtained is exhibited. According to the blood cells or vascular endothelial cells of the present invention, they exhibit high purity and homogenous properties, and are applied to blood for transfusion, a material for treating vascular damage, improving local blood flow, a transplant material, and the like. Shows suitable properties.
  • the manufacturing method of the finger-like cell of this invention the outstanding effect that the finger-like cell which can be differentiated into a blood cell, a vascular endothelial cell, etc. can be efficiently obtained in large quantities is produced.
  • the finger-like cells of the present invention It has an excellent effect of supplying blood cells, vascular endothelial cells, etc. suitable for the purpose of flooring.
  • FIG. 1 shows a lineage diagram of differentiation of hematopoietic stem cells into blood cells.
  • FIG. 2 shows embryonic stem cell colonies 7 days after the start of culture.
  • the scale bar indicates 10 ⁇ m.
  • FIG. 3 shows a partially enlarged view of an embryonic stem cell colony on day 18 after the start of culture.
  • the scale indicates:
  • FIG. 4 shows the ratio of CD34-positive cells.
  • FIG. 5 shows the ratio of CD45-positive cells.
  • FIG. 6 is a diagram showing the morphology of undifferentiated cynomolgus monkey embryonic stem cells and the expression of markers.
  • Panel A shows the results of observation with a phase contrast microscope. In panel A, the scale bar indicates 40 ⁇ m, and in panel A b, the scale bar indicates 40 ⁇ m. Nonel B shows the results of cell surface analysis by FACS.
  • Panel B shows the expression of SSEA-4, a marker of the undifferentiated pluripotency stage (SSEA4 in the figure) (a in panel B), and SSEA-1, a marker of the differentiation stage (SSEA1 in the figure). ”) (In panel B, b).
  • the black line shows the staining result of isotype control IgM or IgG3, and the green line shows the staining with anti-SSEA-1 antibody or anti-SSEA-4 antibody, respectively.
  • FIG. 7 is a diagram showing the results of examining finger-like cell force on blood cells.
  • Panel A shows the results of observation of finger-like cells with a phase contrast microscope.
  • the scale bar indicates 40 m.
  • Panel B shows the results of observation of budding finger-like cells (indicated by arrows) with a phase contrast microscope.
  • the scale bar indicates 40 m.
  • Panel C shows the results of phase contrast microscopy of a large number of hematopoietic progenitor cells generated by confluent culture of finger-like cells.
  • the scale bar indicates 40 m.
  • Panel D is a diagram showing the results of cell surface analysis by FACS.
  • Panel D a shows the expression of CD34, a stem cell marker, and b shows the expression of CD45, a hematopoietic system-specific marker.
  • the black line shows the staining result of isotype control IgG, and the green line shows the staining result of anti-CD34 or anti-CD45 antibody.
  • Panel E shows the results of Wright Giemsa staining of hematopoietic progenitor cells.
  • the scale bar indicates 40 m.
  • FIG. 8 shows the results of cell surface analysis of finger-like cells by FACS.
  • the black line shows the results of staining for the isotype control IgG
  • the green lines show the anti-CD34 antibody, anti-CD45 antibody, anti-VE-forcedherin ("VE-Cadherin” in the figure) antibody, anti-Flk-1 ( In the figure, staining with the “FLK-1”) antibody, anti-c kit antibody, anti-CD133 antibody or anti-CD151 antibody is shown.
  • FIG. 9 is a view showing the results of observation of the repetitive generation of hematopoietic progenitor cells using a phase-contrast microscope.
  • Panel A shows the remaining finger-like cells after trypsinization. Scale bar indicates 40 ⁇ m.
  • Panel B shows the results of observation of the cells after culturing for one week, and
  • Panel C shows the results of culturing for two weeks using a phase contrast microscope. The scale bar indicates 40 ⁇ m.
  • FIG. 10 is a diagram showing the results of examining the differentiation of finger-like cells into vascular endothelial cells.
  • Panel A shows the results of observation of vascular endothelial cells using a phase contrast microscope. The scale bar indicates 40 ⁇ m.
  • Panel B shows the results of cell surface analysis by FACS. In Panel B, a indicates CD34, b indicates CD45, and c indicates expression of an endothelial cell-specific marker, VE-force doherin.
  • the black line shows the staining result of isotype control IgG, and the green line shows the staining with anti-CD34 antibody, anti-CD45 antibody or anti-VE-force doherin antibody.
  • FIG. 11 shows the results obtained by exfoliating the finger-like cells by trypsin treatment, transferring the cells to a new culture dish, and then culturing the cells obtained after culturing in a feeder-free OP9 conditioned medium. It is a figure showing the result of having observed a form.
  • FIG. 12 is a diagram showing the results obtained by culturing finger-like cells obtained by further culturing the cells observed in FIG. 11 under conditions suitable for culturing vascular endothelial cells. is there.
  • FIG. 13 is a diagram showing the results of subjecting cells obtained by culturing finger-like cells under conditions suitable for culturing vascular endothelial cells to vascular structure formation assay.
  • FIG. 14 is a diagram showing the results of subjecting finger-like cells to vasculature formation assays.
  • FIG. 15 is a view showing the results of subjecting undifferentiated embryonic stem cells to vasculature formation assays.
  • FIG. 16 is a diagram showing the results of examining the division of finger-like cells into monocyte Z macrophages.
  • Panel A shows the results of Wright-Giemsa staining.
  • Panel B shows anti-CD14 antibody 5 shows the results of immunostaining using Panel B (a) shows the results using an anti-CD14 antibody, and (b) shows the results using an isotype control antibody as a control.
  • Panel C shows the result of examining the nitro blue tetrazolium reduction activity.
  • Panel C (a) shows the results using finger-like cell-derived cells, and (b) shows the results using HL60 cells as a control.
  • FIG. 17 is a view showing a blast colony.
  • the present invention provides a method for culturing primate embryonic stem cells in a culture vessel coated with an extracellular matrix in the presence of a conditioned medium for stromal cells, so that the blood cells Based on the knowledge of the present inventors that they can be separated. Therefore, according to the present invention, blood cells or vascular endothelium with a high degree of strict control, a differentiation without substantial contamination of foreign animal cells, infection with a virus derived from a foreign animal, etc. It is capable of producing cells, producing blood cells or vascular endothelial cells with high efficiency, producing high-purity blood cells or vascular endothelial cells, etc. Techniques and means for differentiating into vascular endothelial cells can be provided.
  • Examples of the primate embryonic stem cells include, for example, force-null monkey embryonic stem cells [Suemori, H., et al., "Power-Quizal blastocyst-derived embryos produced by IVF or ICSI". Establishment of emoryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI.), Dev. Dynamics ⁇ 222, 273-279 (2001) [Thomson, JA, et al., "Isolation of a primate embryonic stem cell line.”, Proc. Natl. Acad. Sci, USA, Vol. 92, P. 7844-1 p.
  • the present invention relates to a feeder-free separation medium (ie, a feeder-free embryonic stem cell blood fraction Erich medium) comprising a stromal cell conditioned medium.
  • a feeder-free separation medium ie, a feeder-free embryonic stem cell blood fraction Erich medium
  • Erich medium a feeder-free embryonic stem cell blood fraction
  • the feeder-free culture medium of the present invention contains a conditioned medium for stromal cells, it is possible to differentiate primate embryonic stem cells into blood cells without using heterologous animal cells as feeders. It has an excellent effect that it can be done. Therefore, blood cells can be obtained substantially without the contamination of foreign animal cells, infection of foreign animal-derived virus, and the like.
  • the feeder-free culture medium for feeder of the present invention After isolating blood cells obtained by culturing using the feeder-free differentiation medium of the present invention from the culture vessel, adding the new feeder-free differentiation medium, and culturing the cells. By doing so, surprisingly, it has an excellent effect of being able to reproduce blood cells. Therefore, the feeder-free culture medium for feeder of the present invention exhibits an excellent effect that blood cells with excellent reproductivity can be produced.
  • the feeder-free fractionating medium of the present invention since the conditioned medium is contained, it is excellent in that blood cells or vascular endothelial cells can be obtained with high efficiency and high purity. It exerts its effect.
  • the human embryonic stem cells had a high CD34 + CD45 + cell generation rate of 7.5 ⁇ 0.5%]. According to the medium, an excellent effect of being able to obtain cells that are almost 100% positive for CD34 specifically expressed on hematopoietic stem cells is exhibited.
  • vascular endothelial cells have been conventionally separated in a system different from that of blood cells.
  • vascular endothelial cells can be obtained by further culturing finger-like cells generated when embryonic stem cells are differentiated into blood cells in a new culture vessel in a new culture medium without feeder differentiation. If you can do it!
  • the "feeder-free" means that no feeder cell is used, or that no feeder cell is used.
  • the blood cells refer to all blood cells.
  • Examples of the blood cells include, for example, hematopoietic stem cells 0102, lymphoid stem cells 0133, lymphoid stellate cell precursors in the embryonic stem cell force blood cell distribution diagram shown in FIG.
  • Cell 0135 lymphoid adenocyte 0103, T lymphocyte precursor 0104, T cell 0105, B lymphocyte precursor 0106, B cell 0107, plasma cell 0108, NK precursor 0109, NK cell 0110, myeloid stem cell 0134 , Myeloid dendritic cell precursor 0111, myeloid dendritic cell 0112, mast cell progenitor cell 0113, mast cell 0114, basophil progenitor cell 0115, basophil 0116, eosinophil progenitor cell 0117, good Eosinophils 0118, granulocyte macrophage precursor cells 0119, macrophage precursor cells 0120, monocytes 0121, macrophages 0122, osteoclast precursor cells 0123, osteoclasts 0124, neutrophil precursor cells 0125, neutrophils 0126, megakaryocyte Sphere precursor cell 0127, megakaryocyte 0128, Platelets 0129, year erythroid progenitor cells 0130, late erythroid progenitor cells 0131, eryth
  • blood cells include hematopoietic stem cell progenitor cells; hematopoietic stem cell power, and all forms of blood cells present in all the processes of shunting until finally shunting to peripheral blood. It is.
  • the conditioned medium refers to a culture supernatant obtained by culturing cells.
  • the conditioned medium used in the present invention may contain factors and the like produced by the stromal cell line during culture.
  • the stromal cells may be any cell line that supports blood cells.
  • OP9 cell line S17 cell line (mouse bone marrow stromal cell), MS-5 cell line (mouse bone marrow stromal cell) And the like.
  • a mouse fetal yolk sac yolk sac
  • a mouse fetal liver a mouse, and the like may be used in place of the stromal cell line as long as the cells have the ability to support blood cells equivalent to the stromal cell line.
  • Primary cultured cells isolated from hematopoietic tissues such as fetal aorta-mesonephros (aorta-gonad-mesonephros; AGM) and endoderm (para-aortic splanchnopleura; PsP) along the fetal mouse aorta may be used.
  • AGM fetal aorta-mesonephros
  • PsP para-aortic splanchnopleura
  • the type of serum described below may be replaced with other animal serum, for example, poma serum, instead of poma serum, and the concentration of the serum may be changed. You may change it.
  • the culture supernatant is required for the separation of embryonic stem cell force and finger-like cells. However, during the separation of embryonic stem cell force into CD45-positive hematopoietic stem cells, the culture supernatant is used throughout the process. In some cases, it is not always necessary to use the culture supernatant temporarily in the process of dividing finger-like cell force into CD45-positive hematopoietic stem cells.
  • the stromal cells may be irradiated cells or untreated cells.
  • component adjustment can be performed.
  • the rate of proliferation of the stromal cell line is reduced, and the consumption rate of nutrient components contained in a culture solution used for culturing the stromal cell line is reduced. can do.
  • the stromal cell line power is reduced in the amount of waste products to be excreted, and the stromal cell line is produced by the stromal cell line, which is necessary for dividing embryonic stem cells into blood cells or vascular endothelial cells.
  • the dose of the radiation is 40 Gy or more, and preferably 50 Gy or more, from the viewpoint of stably suppressing the growth rate of the stromal cells.
  • the viewpoint of sufficiently exerting the effect of differentiating embryonic stem cells into blood cells and vascular endothelial cells, and the above-mentioned necessity for the differentiation of embryonic stem cells into blood cells or vascular endothelial cells From the viewpoint of sufficiently obtaining factors and the like produced by stromal cells, it is 90 Gy or less, preferably 80 Gy or less, more preferably 70 Gy or less, particularly preferably 60 Gy or less. Is desirable.
  • Irradiation is performed using, for example, an X-ray irradiator [trade name: MBR-1520R-3, manufacturer: Hitachi Medical Corporation (English name: Hitachi Medical Corporation)], a tube voltage of 150 kV, and a tube current of 20 mA. Then, the filter can be set to 0.5AL + 0.1Cu, a culture dish of stromal cells can be set in the sample chamber, and irradiation can be performed.
  • an X-ray irradiator [trade name: MBR-1520R-3, manufacturer: Hitachi Medical Corporation (English name: Hitachi Medical Corporation)]
  • a tube voltage of 150 kV and a tube current of 20 mA.
  • the filter can be set to 0.5AL + 0.1Cu
  • a culture dish of stromal cells can be set in the sample chamber, and irradiation can be performed.
  • the conditioned medium prepared by reducing the growth rate of stromal cells by irradiation, irradiation, or another method capable of controlling the growth rate is used for the feeder-free culture medium of the present invention. May be used.
  • the conditioned medium used in the present invention may be an artificially synthesized conditioned medium having components equivalent to those of the conditioned medium obtained by culturing the stromal cells.
  • a component equivalent to a conditioned medium obtained by culturing stromal cells refers to a component or a derivative thereof contained in the conditioned medium obtained by culturing the stromal cells! Can be identified by analysis by an appropriate means, for example, mass spectrometry, various types of chromatography, NMR and the like.
  • the conditioned medium of the stromal cells includes, for example,
  • step C) a step of removing the cultured cala stromal cells obtained in the step B) Can be obtained. Further, when irradiating the stromal cells, the conditioned medium of the stromal cells,
  • ⁇ ′ irradiating the cells obtained in the step a) with radiation, and culturing the obtained cells in a medium suitable for maintaining stromal cells and embryonic stem cells;
  • OP9 cell culture medium 11 ⁇ composition: ⁇ - ⁇ , 20% by volume Activated fetal serum, ImM j8-mercaptoethanol, 1.6 mM L-glutamine, final concentration 100 U / ml penicillin, final concentration 100 gZml streptomycin ⁇ , OP9 cell culture medium 2-1 ⁇ Composition: ⁇ -MEM, 20 volumes % Heat-inactivated fetal calf serum, 0. ImM ⁇ -mercaptoethanol, ImML glutamine, final concentration lOUZml penicillin, final concentration 10 gZ ml streptomycin ⁇ .
  • the stromal cell culturing condition is a force that can be appropriately set depending on the type of the cell to be used.
  • the conditioned medium for stromal cells is, when OP9 cells are used,
  • OP9 cell culture medium 1-1 or 2-1 on a 10cm culture dish at 37 ° C, 5% by volume CO, subculture every 12 days, -60-70% confluent OP9 cell dish is irradiated with 46 Gy of ⁇ -ray for about 10 minutes, cultured at 37 ° C, 5% by volume CO for 4 hours, and then phosphate-buffered.
  • the dish is supplemented with 10 ml of the OP cell culture medium 1-2 or 2-2, and cultured at 37 ° C. and 5% by volume CO for about 12 hours.
  • the feeder-free culture medium of the present invention comprises, in one embodiment, the conditioned medium, a medium component for maintaining primate embryonic stem cells (eg, a culture solution), and a serum. And a cytokine-free feeder-containing medium.
  • the conditioned medium of such an embodiment may contain an antioxidant agent.
  • the "medium component for maintaining primate embryonic stem cells” may be any ordinary medium used for maintaining primate embryonic stem cells. Examples thereof include Iscove's modified Dulbecco's medium (IMDMZF12).
  • the serum is used to maintain embryonic stem cells.
  • a serum lot check refers to determining compatibility with the embryonic stem cells by performing a culture experiment.
  • a suitable serum lot may be different for each embryonic stem cell line. Further, in the present invention, the same components as those of the serum may be used.
  • the cytodynamic force used in the present invention is not particularly limited as long as it is a factor for dividing embryonic stem cells into blood cells and Z or vascular endothelial cells.
  • G-CSF Granulocyte colony stimulating factor
  • GM-CSF granulocyte macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • EPO erythropoietin
  • TPO thrombopoietin
  • Flt3 Ligand FL
  • interleukin IL
  • VEGF vascular endothelial growth factor
  • BMP bone morphogenetic protein
  • BMP bone morphogenetic protein
  • oncostatin M acidic and basic fibroblast growth factor (acidic FGF, basic FGF), angioboye Chin family (eg, Angiopoietin-1 and
  • Angiopoietin-2) and the like The G-CSF has a function of enhancing neutrophil production.
  • EPO erythropoietin
  • TPO thrombopoetin
  • NK cells natural killer cells
  • the present invention relates to a feeder-free culture system for feeder-free separation, comprising a feeder-free differentiation medium and a culture vessel coated with an extracellular matrix.
  • the feeder-free cultivation system for one part of the present invention is characterized by containing the culture medium for one part of the feeder of the present invention and a culture vessel coated with an extracellular matrix. is there. Therefore, according to the culture system for feeder-free fractionation of the present invention, in the absence of xenogeneic cells, primates that are substantially free from contamination of xenogeneic cells, infection with xenogene-derived viruses, and the like. Embryonic stem cells exerts an excellent effect of being able to differentiate into blood cells with high strict control.
  • Examples of the extracellular matrix include collagen, laminin, fibronectin, hyaluronic acid, and other components that are secreted extracellularly and promote cell adhesion. From the viewpoint of inducing differentiation into blood cells and vascular endothelial cells with high efficiency, collagen is preferred. Examples of the collagen include type IV collagen and the like.
  • the coating of the culture container with the extracellular matrix can be performed by a conventional method.
  • the coating with the extracellular matrix for example, the coating with collagen is formed into a three-dimensional structure, so that the embryonic stem cells can contact the collagen on the lateral and upper surfaces and interact with the collagen. It is thought that information exchange will increase and the survival rate and differentiation rate will improve.
  • the culture vessel may be a vessel usually used for culturing cells.
  • the culture system for feeder-free irrigation of the present invention is obtained from primate embryonic stem cells. It is suitable for use in performing shunting on cells.
  • the primate embryonic stem cells are cultured under a feeder-free medium using the feeder-free differentiation medium of the present invention, and the embryonic stem cells are separated into blood cells.
  • the present invention relates to a feeder-free differentiation method from primate embryonic stem cells to blood cells (hereinafter referred to as a feeder-free blood cell differentiation method).
  • the feeder-free blood cell sorting method of the present invention is characterized in that the feeder-free medium for differentiation of the present invention is used. Therefore, according to the feeder-free blood cell differentiation method of the present invention, since the feeder-free culture medium of the present invention is used, contamination of foreign animal cells, infection of foreign animal-derived virus, and the like can be substantially prevented. Primate embryonic stem cell force exerts an excellent effect of being able to divide into blood cells without any accompanying effect.
  • the feeder-free blood cell sorting method of the present invention is characterized in that the feeder-free medium for differentiation of the present invention and a culture vessel coated with an extracellular matrix are used. There is a unique feature. Therefore, according to the feeder-free blood cell sorting method of the present invention, blood cells can be differentiated from primate embryonic stem cells with high strict control! ⁇ ⁇ Excellent effect.
  • a primate embryonic stem cell is placed in a culture vessel coated with an extracellular matrix, under a feeder-free manner, under the present invention.
  • the cells are cultured in a feeder-free culture medium to generate finger-like cells, and further cultured to generate blood cells.
  • the "finger-like cell” refers to a cell that exhibits a morphology of a finger or joint-like structure, as shown in panel A of FIG.
  • vigorous “finger-like cells” are obtained by starting culturing of undifferentiated primate embryonic stem cells in the feeder-free culture medium of the present invention and then using the force-quizal embryonic stem cells for 1 day.
  • Hematopoietic endothelium has also been shown to have "hemangioblast” activity in mice that are positive for CD151, also known as a marker for vascular endothelial cells.
  • VE-force doherin which is one of the best, is negative and is positioned in primates as a novel cell population that exhibits hemangioblast activity.
  • Finger-like cells also have the characteristics of tissue stem cells, as they are positive for the undifferentiated mesoderm marker Flk-1 and the c kit expressed in immature cells in various tissues. Conceivable.
  • the “heman geoblast” activity refers to the ability of one cell to differentiate into a blood cell line or a vascular endothelial cell line depending on the culture conditions.
  • the present invention is characterized in that primate embryonic stem cells are cultured in a culture vessel coated with an extracellular matrix, under a feeder-free condition, and in the feeder-free fractionating medium. Methods for producing finger-like cells are also included.
  • the present invention also includes vigorous finger-like cells.
  • the finger-like cells of the present invention are obtained by culturing primate animal embryonic stem cells in a culture vessel coated with an extracellular matrix under a feeder-free medium under a feeder-free culture medium of the present invention. Cells.
  • the finger-like cells can be sorted and separated using, for example, the morphology of a finger or joint-like structure, the expression of a marker such as CD151, Flk-1, c kit, VE-force doherin as an index. .
  • a marker such as CD151, Flk-1, c kit, VE-force doherin as an index.
  • cell sorting by flow cytometry using an antibody against the marker for example, CD151, Flk-1, c-kit, VE-cadherin, etc.
  • cell sorting using magnetic beads carrying the antibody can be separated.
  • the finger-like cells allow, for example, blood cells and vascular endothelial cells, which are cells further downstream in the differentiation lineage, to be supplied for clinical purposes and basic research. That is, in the former, not only supply of material as cell therapy for a group of diseases accompanied by hematological diseases and vascular lesions, but also expansion stem cells or myeloid progenitor cells undergo cell fusion in various tissues or cross-transfer in such tissues. Since it is broadly involved in tissue regeneration through transdifferentiation, it is possible to supply an effective material for treatment in regenerative medicine in general.
  • the finger-like cells may be used to express the expression of markers such as VE-forcedherin and CD151, which are markers specific to vascular endothelial cells, for example, by using a specific antibody, a specific probe, a primer pair, or the like. It can be confirmed by showing that VE-force doherin negative CD151 positive.
  • markers such as VE-forcedherin and CD151, which are markers specific to vascular endothelial cells, for example, by using a specific antibody, a specific probe, a primer pair, or the like. It can be confirmed by showing that VE-force doherin negative CD151 positive.
  • the finger-like cells are activated, for example, by culturing them using the above-mentioned "medium suitable for maintaining stromal cells and embryonic stem cells” or a medium to which various cytokins are optionally added. Is amplified.
  • the same culture vessel is replenished with the above-mentioned medium and the culture is continued. During this period, the culture is maintained in the same culture vessel.
  • hematopoietic stem cells or hematopoietic progenitor cells are converted into cells that are positive for hematopoietic system-specific markers CD34 and CD45 (CD34-positive CD45-positive cells). Obtainable.
  • CD34-positive and CD45-positive cells obtained by the feeder-free blood cell sorting method of the present invention exhibit the property of having prominent nucleoli having a high nuclear Z cytoplasmic ratio.
  • colony assays using semi-solid media such as methylcellulose blastocysts were formed with high efficiency (plating efficiency> 80%) (see Fig. 17), and undifferentiated hematopoietic cells with extremely high hematopoietic ability It shows the property as.
  • CD34-positive CD45-positive cells hematopoietic stem cells, lymphoid stem cells, lymphoid dendritic cell precursor cells, lymphoid dendritic cells, T lymphocyte precursor cells, T cells, B lymphocyte precursor cells, B cells, plasma cells, NK progenitor cells, NK cells, myeloid stem cells, myeloid dendritic cell precursor cells, myeloid dendritic cells, mast cell progenitor cells, mast cells, basophil progenitor cells, Basophils, eosinophil precursor cells, eosinophils, granulocyte macrophage precursor cells, macrophage precursor cells, monocytes, macrophages, osteoclast precursor cells, osteoclasts, neutrophil precursor cells, neutrophils It can be divided into cells such as megakaryocyte precursor cells, megakaryocytes, platelets, early erythroid precursor cells, late erythroid precursor cells, and erythrocytes.
  • the undifferentiated embryonic stem cells at the center of the colony of embryonic stem cells maintained in an undifferentiated state on a culture dish are removed. If the population is removed and a 6 ⁇ eldish dish coated with type IV collagen is used as the culture vessel coated with extracellular matrix, 1 x 10 1 — 1 x 10 3 cells per 1 ml It is desirable that the cells are further seeded so as to have 1 ⁇ 10 2 cells.
  • the conditions for culturing primate embryonic stem cells in a feeder-free culture medium can be appropriately set according to the type of primate embryonic stem cells used. Conditions such as volume% CO are exemplified.
  • the medium may be appropriately replaced with a new feeder-free medium for culture according to the form and number of cells derived from primate embryonic stem cells.
  • the finger-like cells are transformed several days after the start of cultivation, for example, about 3 days in the case of virulent monkey embryonic stem cells. After being generated and confluent, circular cells are generated from the finger-like cells.
  • the circular cells obtained by the feeder-free blood cell sorting method of the present invention can be used to express, for example, the expression of markers such as stem cell marker CD34 and hematopoietic system-specific marker CD45. It can be confirmed by examining by a conventional method using a specific antibody, a specific probe, a primer pair and the like, and showing CD34 positive and CD45 positive.
  • primate embryonic stem cells are cultured in a feeder-free differentiation medium of the present invention in a culture vessel coated with an extracellular matrix under a feeder-free medium, Generating finger-like cells, transferring the finger-like cells to a culture vessel containing a fresh medium and coated with an extracellular matrix, and further culturing to generate vascular endothelial cells.
  • a feeder-free differentiation method from primate embryonic stem cells to vascular endothelial cells hereinafter referred to as a feeder-free vascular endothelial cell differentiation method).
  • the feeder-free vascular endothelial cell sorting method of the present invention is characterized in that the feeder-free medium for vascular endothelial cells of the present invention is used. Therefore, the fee-free of the present invention According to the vascular endothelial cell differentiation method, primate embryonic stem cell force can be differentiated into vascular endothelial cells without substantially contaminating foreign animal cells, infecting foreign animal-derived viruses, etc. It has an excellent effect.
  • steps up to the generation of finger-like cells are the same as in the feeder-free blood cell differentiation method.
  • Separation of finger-like cells from a culture vessel coated with an extracellular matrix can be performed by a conventional method used for detachment of cells from a culture vessel, for example, trypsin treatment.
  • the separated finger-like cells are transferred to a culture vessel coated with an extracellular matrix, which contains a new feeder-free separation medium, and is further cultured. .
  • the culture conditions for the finger-like cells may be the same as the culture conditions for the embryonic stem cells in the feeder-free blood cell differentiation method, for example, conditions of 37 ° C and 5% by volume CO.
  • vascular endothelial cells are negative for hematopoietic system-specific markers CD34 and CD45 and positive for endothelial cell-specific marker VE-cadherin.
  • CD34-negative, CD45-negative VE-cadherin-positive cells CD34-negative, CD45-negative VE-cadherin-positive cells.
  • the CD34-negative CD45-negative VE-force doherin-positive cells have a cobblestone shape.
  • the CD34-negative CD45-negative VE-force doherin-positive cells have excellent properties that can be passaged for several months in a new dish and can be stably amplified.
  • the CD34-negative CD45-negative VE-forced-herin-positive cells exhibiting the cobblestone shape are, for example, cells that can be passaged for several months in OP9-conditioned medium containing cytoforce. Yes, self-amplification is possible in vitro. Therefore, the use of powerful cells provides an excellent effect that large-scale preparation of a transplant is possible. Further, by culturing the CD34-negative CD45-negative VE-forced-herin-positive cells exhibiting the cobblestone shape in a medium suitable for culturing vascular endothelium, it is possible to obtain cord-like structural cells forming a vascular lumen. it can.
  • the cord-like cells forming such a blood vessel lumen are useful, for example, for preparing functional cells immediately before transplantation.
  • the vascular endothelial cells obtained by the feeder-free vascular endothelial cell sorting method of the present invention include, for example, CD34 and CD45, which are hematopoietic system-specific markers, and VE-forcedherin, which is an endothelial cell-specific marker.
  • CD34 and CD45 which are hematopoietic system-specific markers
  • VE-forcedherin which is an endothelial cell-specific marker.
  • the expression of such a marker can be confirmed by, for example, examining the expression of a marker using a conventional method using a specific antibody, a specific probe, a primer pair and the like, and showing CD34 negative CD45 negative VE-dherin positive.
  • the present invention provides a method for culturing a primate embryonic stem cell in a culture vessel coated with an extracellular matrix under a feeder-free medium under a feeder-free differentiation medium of the present invention.
  • the present invention relates to a method for producing blood cells, characterized in that the embryonic stem cells are differentiated into blood cells, and then the blood cells are detached and isolated.
  • the method for producing blood cells of the present invention has one significant feature in that the feeder-free culture medium of the present invention is used. Therefore, according to the method for producing blood cells of the present invention, it is possible to obtain high-purity blood cells without substantially contaminating foreign animal cells, infecting foreign animal-derived viruses, and the like. Demonstrate.
  • the method for producing blood cells of the present invention is based on the fact that primate embryonic stem cells are cultured in a culture vessel coated with an extracellular matrix in a feeder-free culture medium of the present invention. There is one major feature. Therefore, according to the method for producing blood cells of the present invention, if primate embryonic stem cells can be differentiated into vascular endothelial cells with a high degree of strict control, an excellent effect is exhibited.
  • the culture medium for feeder-free separation of the present invention is added to the culture vessel after blood cell separation, and the culture medium is further cultured under the feeder-free state. And a method for obtaining blood cells.
  • the feeder-free medium of the present invention is added to the culture vessel after the blood cells are detached, and the culture is further performed under the feeder-free.
  • the culture is further performed under the feeder-free. Therefore, blood cells can be obtained. Therefore, according to the method for producing blood cells of the present invention, an excellent effect that blood cells can be obtained with excellent reproductivity over a long period of time can be obtained.
  • the method for producing blood cells of the present invention can be carried out by the same procedure as the feeder-free blood cell sorting method of the present invention.
  • a feeder-free method that allows CD34-positive and CD45-positive cells to be further differentiated under appropriate conditions in accordance with the type of target blood cells.
  • the cytodynamic force in the medium for one-part dani may be appropriately changed.
  • examples of production of blood cells by various cytokins include differentiation into granulocytes by G-CSF and GM CSF, and monocyte Z macrophages by GM CSF and M CSF. Differentiation, differentiation into NK cells by IL-15, differentiation into erythrocytes by EPO, differentiation into megakaryocyte Z platelets by TPO, differentiation into dendritic cells by IL4 and GM-CSF, and the like.
  • the target blood cells are sorted using, for example, the expression of markers specific to the blood cells, for example, markers such as CD34 and CD45, as an index.
  • markers specific to the blood cells for example, markers such as CD34 and CD45
  • target blood cells are separated by cell sorting by flow cytometry using an antibody against the above-mentioned marker, for example, CD34, CD45, etc., cell sorting using magnetic beads holding the antibody, etc. can do.
  • the stromal cell force of a fibroblast cell line such as OP9 cells is obtained.
  • Macrophages can be produced by culturing the cells in a feeder-free medium, which has also obtained the stromal cell strength of a bone marrow cell line such as 5 cells.
  • Blood cells obtained by a powerful production method are also included in the present invention.
  • the blood cells of the present invention are obtained by the production method of the present invention, they exhibit excellent properties such that there is substantially no contamination with foreign animal cells, infection with foreign animal-derived viruses, and the like. Further, since the blood cells of the present invention are obtained by the production method of the present invention, they exhibit high purity and uniform properties. Therefore, the blood cells of the present invention can be used for blood for blood transfusion, for use in the production of blood for blood transfusion, and as materials for basic research relating to hematopoietic mechanisms.
  • the blood cells of the present invention can be maintained under a nitrogen gas freezing condition, for example, in a medium such as a cell cryopreservation liquid such as a cell banker (manufactured by Juji Kagaku).
  • a medium such as a cell cryopreservation liquid such as a cell banker (manufactured by Juji Kagaku).
  • the present invention provides that primate embryonic stem cells are coated with an extracellular matrix. Culturing in a feeder-free differentiation medium of the present invention under a feeder-free culture medium in a culture vessel prepared to generate finger-like cells, and the finger-like cells are filled with a new medium and coated with an extracellular matrix.
  • a method for producing vascular endothelial cells which comprises transferring the embryonic stem cells to vascular endothelial cells and isolating the vascular endothelial cells. .
  • the method for producing vascular endothelial cells of the present invention comprises culturing primate embryonic stem cells in a feeder-free differentiation medium of the present invention in a culture vessel coated with extracellular matrix, under a feeder-free condition. There is one major feature of generating finger-like cells. Therefore, according to the method for producing vascular endothelial cells of the present invention, it is possible to obtain vascular endothelial cells substantially free from contamination with foreign animal cells, infection with foreign animal-derived virus, and the like.
  • the method for producing vascular endothelial cells of the present invention has one great feature in that finger-like cells are transferred to a culture vessel containing a new medium and coated with an extracellular matrix for further culturing. . Therefore, according to the method for producing vascular endothelial cells of the present invention, it is surprising that vascular endothelial cells can be surprisingly obtained by high-precision control, and high-purity vascular endothelial cells can be obtained! / ⁇ ⁇ Excellent effect.
  • vascular endothelial cells can be separated and sorted using, for example, the expression of a marker such as VE-drugin or PECAM (CD31) as an index.
  • vascular endothelial cells are separated by cell sorting by flow cytometry using an antibody against a marker such as VE-cadherin or PECAM (CD31), cell sorting using magnetic beads holding the antibody, or the like. can do.
  • Vascular endothelial cells obtained by a powerful production method are also included in the present invention.
  • the vascular endothelial cell of the present invention since it is obtained by the production method of the present invention, it is excellent in that there is substantially no contamination with foreign animal cells, no infection with foreign animal-derived virus, and the like. Show properties.
  • the vascular endothelial cells of the present invention are obtained by the production method of the present invention, and thus exhibit high purity and uniform properties.
  • vascular endothelial cell of the present invention a material for treating vascular injury or improving local blood flow, a transplant material, a use for producing these materials, and a method for treating vascular endothelium. It can be used as a material in basic research on the mechanism of development and differentiation.
  • vascular endothelial cell of the present invention it is possible to obtain a three-dimensional vascular structure by, for example, culturing in a collagen gel or the like.
  • the vascular endothelial cells of the present invention can be maintained under a nitrogen gas freezing condition, for example, in a medium such as a cell banker (manufactured by Juji Kagaku Co., Ltd.) dedicated to cell freezing and storage.
  • a medium such as a cell banker (manufactured by Juji Kagaku Co., Ltd.) dedicated to cell freezing and storage.
  • the feeder-free culture medium for fractionation and the filtration method using the same according to the present invention are limited to the technique for fractionating embryonic stem cells into blood. Depending on the type of inn, it can be applied and developed as a technique for dividing into various cells.
  • OP9 cells which are stromal cells, were cultured in an OP9 cell culture medium 1-1 ⁇ composition: ⁇ -MEM (manufactured by Invitrogen Corp.), 20% by volume of heat-inactivated ⁇ fetal serum [P. Mbenow (PAA Laboratories GmbH)], lmM j8—mercaptoethanol (manufactured by Sigma Chemical Co.), 1.6 mM
  • OP9 cells were subcultured and maintained about every three days so as not to become confluent. Specifically, the OP9 cell culture medium of Zabu confluent was removed, and reacted with 0.25% by weight of trypsin-HBSS (Hanks balanced salt solution) solution for about 2 minutes to detach the cells. After washing the cells with the medium, reduce the cell density by a factor of several and resuspend the cells in the medium. The cells were sowing diameter 10cm culture dish, in a CO 2 incubator, and cultured at 37 ° C, 5 volume 0/0 CO.
  • trypsin-HBSS Hops balanced salt solution
  • the culture dish was irradiated with radiation (150 kV, 20 mA, 60 Gv, 10 minutes) before becoming confluent. Then, in the culture dish, 0.25 weight 0/0 Bok The cells were peeled off while adding lysin HBSS (Hanks balanced salt solution) solution and pipetting. The trypsin solution was removed from the solution containing the obtained cells, and the OP9 cell culture medium 1-2 ⁇ composition: Iscove's modified Dulbecco's medium (IMDM) [Sigma Chemical Co., Ltd.
  • IMDM Iscove's modified Dulbecco's medium
  • the cells were washed once with L-glutamine (manufactured by Invitrogen Corp.) ⁇ . Thereafter, the obtained cells were suspended in the OP9 cell culture medium 12 described above. The resulting cell suspension is placed on a 6-well flat-bottom multiwell plate at 3 x 10 4 cells per well and placed in a CO incubator.
  • undifferentiated primate embryonic stem cells undifferentiated monkey embryonic stem cells passaged from primary fibroblasts prepared from mouse embryos as a feeder cell were used. 0.25% by weight of trypsin HBSS solution was added to the culture dish of the undifferentiated sal embryonic stem cells, and the cells were peeled off. OP9 conditioned medium 1, final concentration 20 ngZml vascular endothelial growth factor (VEGF), final concentration 20 ngZml bone morphogenetic protein 4 (BMP-4), 20 ng stem cell factor (SCF), final concentration lOngZml Flt3-ligand ⁇ . The obtained cell suspension was seeded on a collagen-coated plate (Betaton Dickinson, 6-well multiwell plate coated with mouse type IV collagen). Then, the embryonic stem cells were placed in a CO incubator at 37 ° C and 5 volumes.
  • VEGF vascular endothelial growth factor
  • BMP-4 bone morphogenetic protein 4
  • SCF stem cell factor
  • FIG. 2 shows colonies of embryonic stem cell-derived cells on day 7 after the start of culture.
  • culture On the 7th day after the start, the culture medium was changed to a medium for inducing induction 1 2 ⁇ Composition: The above-mentioned feeder-free medium for OP9 conditioned medium 1, final concentration 20 ngZml VEGF, final concentration 20 ngZml BMP-4, final concentration 20 ⁇ g / ml SCF, final concentration 20ngZml Flt3—ligand, final concentration lOngZml interleukin 6 (IL-6), final concentration IngZml interleukin 3 (IL-3), final concentration IngZml granulocyte macrophage colony stimulating factor (GM—CSF) ⁇ And the culture was continued.
  • IL-6 lOngZml interleukin 6
  • IL-3 IngZml interleukin 3
  • GM—CSF granulocyte macrophage colony stimulating factor
  • the medium 12 was replaced with the differentiation-inducing medium 13 ⁇ the feeder-free differentiation-free OP9-conditioned medium 1, the final concentration of 20ngZml SCF, the final concentration of 20ngZml Flt3-ligand, the final concentration of lOngZml IL— 6.
  • the final concentration was replaced with IngZml IL-3 and the final concentration IngZml GM-CSF, and the culture was continued.
  • the culture dish 0.1 to ⁇ Ka ⁇ 25 weight 0/0 trypsin solution lml, while vigorously piperidines ting, by incubation at 37 ° C, the cells were harvested. The collected cells were washed with phosphate buffered saline (PBS). Then, 1 ⁇ 10 6 cells were incubated on ice for 30 minutes for primary antibodies against markers such as CD34 and CD45, ie, PE-conjugated anti-CD34 antibody (BD Biosciences) or FITC-conjugated anti-CD45 antibody. [BD Biosciences]. Thereafter, the expression level of each marker was analyzed using FACSCalibur (trade name, manufactured by BD Biosciences).
  • FACSCalibur trade name, manufactured by BD Biosciences.
  • FIG. 4 and FIG. 5 show the results.
  • FIG. 4 shows the ratio of CD34-positive cells.
  • FIG. 5 shows the ratio of CD45-positive cells.
  • CD45-positive cells were 70.60%, and CD34-positive cells were 92.60%. On average, CD45-positive cells accounted for 30-50%, and CD34-positive cells accounted for 70-90%.
  • OP9-conditioned medium for feeder-free one-part dani 2 The OP9 cells were cultured in an OP9 cell culture medium 2-1 ⁇ composition: ⁇ -MEM (manufactured by Invitrogen Corp.), 20% by volume of a selected lot of heat-inactivated ⁇ fetal serum [P. PAA Laboratories GmbH], 0. ImM j8-mercaptoethanol (manufactured by Sigma Chemical Co.), ImM L-glutamine (manufactured by Invitrogen Corp.), final concentration lOUZml ⁇ - A final concentration of 10 g Zml streptomycin [Invitrogen Corp.] ⁇ was added to the sample on a 10 cm culture dish.
  • the culture supernatant on the culture dish was aspirated, and 0.2% by weight trypsin Z0.02% by weight EDTA solution [Gibco (GIBCO) was added to the cells on the culture dish. CO), catalog number: 23200-072] 2 ml was added to the mixture and incubated at 37 ° C for 2-3 minutes. Thereafter, 2 to 18 ml of a new culture medium for OP9 cell culture was added to the suspension and well suspended. The resulting suspension was centrifuged to remove the supernatant. Thereafter, the OP9 cells were suspended in a new OP9 cell culture medium 2 to 110 ml so that the cell concentration of the culture was about 1Z3-1Z6. Transfer the resulting cell suspension onto a 10 cm culture dish,
  • a dish of OP9 cells that had become 60-70% confluent in a 10-cm culture dish was placed on the focus table surface of the product name: MBR-1520R-3 (manufactured by Hitachi Medical) at 290 mm, and 46 Gy gamma rays were used. For 4 hours. Thereafter, 9 cells were washed with phosphate buffered saline ( ⁇ S), and 10 ml of ⁇ cell culture medium 2-2 ⁇ composition: Iscove's modified Dulbecco's medium (IMDM) [Invitrogen Corp. )], 15% by weight of selected lot, heat-inactivated fetal serum [PAA Laboratories GmbH], 0.
  • IMDM Iscove's modified Dulbecco's medium
  • ImM j8-menolecaptoethanol [Sigma Chemical Co.] 3 mM L-glutamine (manufactured by Invitrogen Corp.), 5 M hydrocortisone, final concentration lOUZml ⁇ -sylin (manufactured by Invitrogen Corp., final concentration 10 gZml Streptomycin [Invitrogen Corp.] Manufactured). Then, OP9 cells In a CO 2 incubator, 37 ° C, 5 volume 0/0 CO in cultured for 12 hours, th 1st
  • the supernatant of the culture was collected.
  • Fetal mice at fetal age of 12.5-13.5 were removed from the uterus, and the brain, limbs, internal organs (gastrointestinal tract, liver, kidney, lung) and tail were removed, and only the trunk tissue was recovered. After that, the obtained trunk tissue was cut finely with dissecting scissors, and then the inside of the 18G needle was moved up and down several times to further divide it. The obtained divided tissue was transferred to a 50 ml tube containing 5 ml of glass beads, and 10 ml of a 0.25% by weight trypsin solution was further added. Thereafter, the divided tissues were incubated at 37 ° C with stirring every 10 minutes.
  • DMEM fetal bovine serum
  • a cell suspension was collected.
  • the cell suspension was passed through a sterile mesh (40 m pore, manufactured by BD Falcon, trade name: Cell Strainer) to remove cell aggregates and obtain a cell stock.
  • the obtained cells were frozen and stored as a frozen cell stock.
  • the frozen cell stock was thawed and cultured in a DMEM medium containing 10% by volume of fetal serum at 37 ° C and 5% by volume of CO. After one passage, when it becomes confluent,
  • Mitomycin C (manufactured by Sigma) was added to a final concentration of lOmgZl, and the mixture was incubated at 37 ° C for 3 hours. Thereafter, the cells were washed three times with PBS and collected to obtain mitomycin C-treated embryonic fibroblasts.
  • the obtained mitomycin C-treated embryonic fibroblasts were seeded on a 6 cm dish at 1 ⁇ 10 6 cells per dish, and then inoculated in a CO incubator at 37 ° C.
  • Kakar MEF The culture of Kakar MEF should be started at least 8 hours before seeding embryonic stem cells. did.
  • the force-quiz monkey embryonic stem cell line CMK-6 was used as a primate.
  • the supernatant was removed from the force-quizal embryonic stem cell culture dish, and the embryonic stem cells were washed once with PBS.
  • 1 ml of 0.25% by weight trypsin solution was added, and the embryonic stem cells were incubated at 37 ° C for 1 minute. Immediately thereafter, the side of the dish was tapped to suspend embryonic stem cells.
  • the suspended embryonic stem cells were added to a medium for maintaining undifferentiated embryonic stem cells ⁇ composition: MEMZF12 medium (manufactured by Invitrogen Corp.)], and 20% by volume heat-inactivated ⁇ fetal serum of the selected lot.
  • FBS (PAA Laboratories GmbH)
  • bFGF basic fibroblast growth factor
  • BMP-4 final concentration lOngZml bone formation Protein
  • LIF U / ml leukemia inhibitory factor
  • ImM j8- Mercaptoethanol manufactured by Sigma Chemical Co.
  • ImML-glutamine manufactured by Invitrogen Corp.
  • final concentration lOUZml penicillin manufactured by Invitrogen Corp.
  • the embryonic stem cells were collected in another tube.
  • the cells were well suspended in the embryonic stem cell undifferentiated maintenance medium, centrifuged, and the supernatant was removed.
  • the obtained cells were suspended in 20 ml of a new medium for maintaining undifferentiated embryonic stem cells.
  • the obtained embryonic stem cell suspension was placed on the mouse embryonic fibroblasts of the MEF dish for maintaining undifferentiated embryonic stem cells obtained in (1) above, in 5 ml portions in a single dish. Seeds on a single dish and incubate at 37 ° C, 5% CO in a CO incubator.
  • the cells were directly observed with a phase-contrast optical microscope (Olympus Corporation, trade name: 1X70). did.
  • the cells were examined for expression of markers such as SSEA-1 and SSEA-4 on the cell surface as follows. After the culture was completed, lml of 0.25% by weight trypsin solution was added to the dish, and the dish was incubated at 37 ° C for 1 minute. Then, the side of the dish was immediately hit to detach only embryonic stem cells. The collected cells were washed with an isotonic solution containing a bivalent ion chelating agent such as EDTA and at the same time the cells were loosened by pipetting.
  • markers such as SSEA-1 and SSEA-4
  • the cultured cells formed flat circular colonies having a polygonal cytoplasm and a single nucleus having a large nucleus.
  • the cells exhibited high levels of SSEA-4 and low levels of SSEA-1. Therefore, it was considered that most of the cells after culturing were maintained in an undifferentiated state.
  • the inside of the embryonic stem cell colony on the second day of the passage was roughly cut with a pasteur pipe (fine capillary) having a thin tip. Then, using a fine capillary, a population of unsegregated embryonic stem cells at the center of the colony was aspirated and removed under an optical microscope. As a result, contamination of mouse embryonic fibroblasts was substantially eliminated.
  • differentiation-inducing medium 2 ⁇ composition OP9-conditioned medium 2 for feeder-free differentiation, final concentration
  • VEGF vascular endothelial growth factor
  • SCF cell factor
  • lOngZml Flt3-ligand final concentration lOngZml IL-6
  • 20 ngZml IL-3 ⁇ was added to type IV collagen coat-6 ⁇ Eldish [BD Biosciences]
  • a culture dish was prepared in advance by adding the cells to be used for inducing differentiation of embryonic stem cells.
  • the undivided embryonic stem cell population was gently pipetted to partially loosen the culture dish, and the number of selected ES colonies was reduced to 1 ⁇ 10 2 per square well. Seeded. Thereafter, the embryonic stem cells were placed in a CO incubator at 37 ° C.
  • the cells were cultured in 5% by volume of CO to induce differentiation. Change the medium every 3-4 times
  • CD34 is a stem cell marker of various tissues such as hematopoietic cells, nerve cells, endothelial cells, etc. It was positive for the specific marker CD45.
  • FIG. 7 panel E the cells were shown to have prominent nucleoli with a high nuclear Z cytoplasmic ratio, indicating immature hematopoiesis. Characteristics of cells It matched the sign. Thus, almost all of the round cells were CD34-positive and CD45-positive, indicating that they were hematopoietic stem cells or hematopoietic progenitor cells.
  • Flk-1 which is a marker for immature mesoderm
  • CD34 and C kit which are markers for immature cells including hematopoietic stem cells
  • specific markers for blood cells in general was reacted with a primary antibody against a marker such as CD45, a vascular endothelial cell-specific marker, VE-forcedherin, and a vascular endothelial marker, CD151.
  • the secondary antibody reaction was continued.
  • the expression level of each marker was analyzed using FACSCalibur (trade name, manufactured by BD Biosciences).
  • PE-conjugated anti-CD34 antibody manufactured by BD Biosciences
  • FITC-conjugated anti-CD45 antibody manufactured by BD Biosciences
  • PE-conjugated anti-VE-forcedherin antibody [ Chemicon International, Inc.), PE-conjugated anti-CD 133 antibody (Miltenyi Biotech GmbH), PE-conjugated anti-Flk-1 antibody (BD Biosciences) Manufactured
  • PE-conjugated anti-c kit (CD117) antibody manufactured by BD Biosciences
  • anti-CD151 antibody manufactured by BD Biosciences
  • the “finger-like cells” were VE-active doherin-negative Flk-1 positive c kit-positive CD151-positive.
  • Example 4 (3) At 7-10 days after the differentiation induction in Example 4 (3), 0.25% by weight of trypsin solution was added to the culture dish containing the confluent finger-like cells, and pipetting was performed at room temperature. The finger-like cells were exfoliated while doing so.
  • Example 3 In Example 3 described above, at a stage 7 to 10 days after the induction of the shroud, a 0.25% by weight trypsin solution was added to the culture dish, and the finger-like cells were exfoliated while pipetting at room temperature. To the obtained finger-like cells, 22 ml of the OP-conditioned medium for feeder-free differentiation described in Example 3 above was added and sufficiently suspended. Then, the supernatant was removed by centrifugation, and the above-mentioned “medium suitable for culturing stoma cells and embryonic stem cells” was diluted so that the original cell density was about 1 Z3-1Z6. .
  • the obtained cell dilution was cultured on a new 6 ⁇ Eldish or collagen-coated 6 ⁇ Eldish at 37 ° C and 5% by volume CO.
  • the cobblestone cells were treated with a 0.02% by weight EDTA solution and collected, and the collected cells were Washed with PBS. Thereafter, 1 ⁇ 10 6 cells are allowed to react on ice for 30 minutes with a primary antibody against a marker 1 such as hematopoietic system-specific markers CD34 and CD45 and an endothelial cell-specific marker VE-forcedherin. The secondary antibody reaction was continued. Subsequently, the expression level of each marker was analyzed using FACSCalibur (trade name, manufactured by BD Biosciences).
  • a PE-conjugated anti-CD34 antibody manufactured by BD Biosciences
  • a FITC-conjugated anti-CD45 antibody manufactured by BD Biosciences
  • an anti-VE antibody [Chemicon International, Inc. (Chemicon International, Inc.)] was used.
  • the cobblestone cells were CD34-negative, CD45-negative VE-force doherin-positive. Therefore, it was suggested that the finger-like cells were differentiated into vascular endothelial cells. In addition, the cobblestone cells proliferated vigorously in vitro, could be passaged for several months in a new dish, and could be expanded stably and with good reproducibility.
  • Example 4 The finger-like cells obtained in Example 4 were also peeled off the dish force using trypsin ZEDTA (manufactured by GIBCO-BRL). Then, the obtained cells (0.5 ⁇ 10 6 —1 ⁇ 10 6 cells per square well) were placed on a 6-well flat bottom multi-well plate, and the OP9 conditioned medium for feeder-free fractionation described in Example 3 above was used. 22.5 ml or a medium suitable for culturing vascular endothelium [trade name: EGM-2 BulletKit (Code B3162), manufactured by Takara Bio Inc.] 2. Cultured in 5 ml at 37 ° C, 5% by volume CO for 3 days. Transfer the obtained cells to the phase
  • a PE-conjugated anti-VE-force doherin antibody which is an antibody against VE-force doherin which is a vascular endothelial cell-specific marker [Chemicon International, Inc. was used to determine the expression of VE-force doherin [0159]
  • it was positive for VE-force doherin indicating that it was distributed to vascular endothelial cells.
  • Example 7 In order to confirm that the cells obtained in Example 7 were endothelial cells, a vasculature formation assay was performed as follows.
  • Example 7 the product name: EGM-2 BulletKit [manufactured by Takara Bio Inc., Code
  • the product name: EGM-2 BulletKit [Takara Bio Inc. , Code B3162] the vascular endothelial cells obtained from the vascular endothelial cells were found to form a vasculature when subjected to a vasculature formation assay.
  • vasculature was partially formed, but they formed colonies and formed clumps. You can see that there are some that are. Therefore, it is suggested that the obtained cells are in an undivided stage compared to the mature vascular endothelium. Further, as shown in FIG. 15, when the undifferentiated embryonic stem cells were subjected to vasculature formation Atsusei, the formation of vasculature was almost invisible.
  • MS-5 cells a mouse bone marrow stromal cell line [purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ)], were selected from a selected lot of 10% heat-inactivated FBS [PAA Laboratories (PAA Laboratories). GmbH), 2 mM L-glutamine (Invitorgen Corp.), 2 mM sodium pyruvate (Invitorgen Corp.), and lOUZml penicillin (Invitrogen Corp.) And 10 g / ml streptomycin [Invitrogen Corp.], and maintained in a-MEM [Invitorgen Corp .;]. At subconfluence, these cells were treated with 46 Gy of ⁇ -irradiation. After washing with PBS, cells were harvested from selected lots of 15% heat-inactivated FBS (PAA Laboratories GmbH) and 0.1 lm Mj8-mercaptoethanol [Sigma Chemical Co.]. ] And 3 mM
  • the cells were positive for CD14, a component of the LPS receptor complex known as monocyte-Z macrophage-specific promoter, and were divided into macrophages. It was suggested that you do it.
  • the cells were subjected to a reduction assay of troblue tetrazolium salt (NBT) exhibiting active oxygen production activity, and the macrophage function was analyzed.
  • NBT troblue tetrazolium salt
  • 5 ⁇ 10 5 cells were collected, washed once with PBS, and suspended in 1 ml of differentiation-inducing medium. Then, 1 ml of NBT ⁇ solution [lmg NBT (manufactured by Nacalai Tester) Zml differentiation induction medium] was added, and the cells were incubated for 25 minutes in the presence of TPA 100 ng. Thereafter, the cells were washed with PBS and resuspended in 10 ⁇ l of PBS.
  • NBT troblue tetrazolium salt
  • the obtained cell suspension was dropped on a slide glass and covered with a cover glass. ⁇ The number of positive cells was counted using an optical microscope (Olinos, trade name: # 51). As a negative control, human leukemia cells HL-60 cells were used, and a similar reduction assay was performed. The results are shown in panel C of FIG.
  • the present invention it becomes possible to stably provide blood cells and vascular endothelial cells suitable for blood for transfusion, materials for transplantation, and the like on an industrial scale. Further, since the blood cells and the like of the present invention also lead to enhancement of natural healing power, their effects on medical treatment and the medical industry are enormous. Furthermore, if the production of safe and secure transfusion blood instead of the current blood donation is included, there is a possibility of expanding into the huge plant industry.

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Abstract

Il est prévu de fournir un moyen grâce auquel des cellules souches embryonnaires de primate peuvent être différenciées en cellules sanguines ou en cellules endothéliales vasculaires pratiquement exemptes de contamination avec des cellules xénogéniques animales, d'infection avec un virus trouvant son origine dans un animal xénogénique etc. À savoir, un milieu en vue d'une différenciation exempte d'alimentateur qui contient un milieu d'adaptation à des cellules de stroma, un système de culture destiné à une différenciation exempte d'alimentateur qui comprend le milieu ci-dessus en vue d'une différenciation exempte d'alimentateur et un récipient de culture revêtu d'une matrice extracellulaire, un procédé destiné à une différenciation exempte d'alimentateur à partir de cellules souches embryonnaires de primate en cellules sanguines, un procédé destiné à une différenciation exempte d'alimentateur à partir de cellules souches embryonnaires de primate en cellules endothéliales vasculaires, un procédé destiné à la production de cellules sanguines, un procédé destiné à la production de cellules endothéliales vasculaires est un procédé destiné à la production de cellules en forme de doigts utilisant chacune le milieu ci-dessus en vue d'une différenciation exempte d'alimentateur, et des cellules sanguines, des cellules endothéliales vasculaires et des cellules en forme de doigts obtenues par ces procédés.
PCT/JP2005/003474 2004-03-04 2005-03-02 Milieu en vue d'une différenciation exempte d'alimentateur et procédé de différenciation exempt d'alimentateur à partir d'une cellule souche embryonnaire de primate Ceased WO2005085426A1 (fr)

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WO2008056779A1 (fr) * 2006-11-09 2008-05-15 Japan As Represented By The President Of International Medical Center Of Japan Procédé destiné à la culture et au passage d'une cellule souche embryonnaire de primate, et procédé destiné à induire la différenciation de la cellule souche embryonnaire
US20090222936A1 (en) * 2005-11-08 2009-09-03 Timothy Richmond Recombinant Expression of Multiprotein Complexes Using Polygenes
JP2014102147A (ja) * 2012-11-20 2014-06-05 Dastech Inc 生体試料の切断装置及び切断方法並びに細胞観察方法
JP2016119910A (ja) * 2009-12-04 2016-07-07 ステム セル アンド リジェネレイティブ メディスン インターナショナル， インコーポレイテッド ヒト胚性幹細胞由来血管芽細胞からナチュラルキラー細胞および樹状細胞を生成する方法

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US20090222936A1 (en) * 2005-11-08 2009-09-03 Timothy Richmond Recombinant Expression of Multiprotein Complexes Using Polygenes
US9512443B2 (en) * 2005-11-08 2016-12-06 Eth Zurich Recombinant expression of multiprotein complexes using polygenes
WO2008056779A1 (fr) * 2006-11-09 2008-05-15 Japan As Represented By The President Of International Medical Center Of Japan Procédé destiné à la culture et au passage d'une cellule souche embryonnaire de primate, et procédé destiné à induire la différenciation de la cellule souche embryonnaire
JP2016119910A (ja) * 2009-12-04 2016-07-07 ステム セル アンド リジェネレイティブ メディスン インターナショナル， インコーポレイテッド ヒト胚性幹細胞由来血管芽細胞からナチュラルキラー細胞および樹状細胞を生成する方法
JP2019083819A (ja) * 2009-12-04 2019-06-06 ステム セル アンド リジェネレイティブ メディスン インターナショナル， インコーポレイテッド ヒト胚性幹細胞由来血管芽細胞からナチュラルキラー細胞および樹状細胞を生成する方法
JP2014102147A (ja) * 2012-11-20 2014-06-05 Dastech Inc 生体試料の切断装置及び切断方法並びに細胞観察方法

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